The long-term objective of this proposed research is to characterize the molecules and mechanisms involved in regulating mitotic spindle structure and function. Recent work from our lab has revealed that the unconventional myosin, Myosin-10 (Myo10), localizes to the spindle, and reduction of Myo10 protein levels in cells produces several spindle phenotypes. Preliminary data confirm a direct interaction between Myo10 and the major cell cycle regulatory kinase, Wee1, as well as suggest a functional interaction between the two proteins in regulation of the mitotic spindle. In order to examine the hypothesis that Myo10 associates with Weel1during mitosis in the regulation of mitotic spindle structure and function, the following Specific Aims will be addressed using the Xenopus laevis embryo system: (1) embryo extracts and a series of biochemical techniques will be used to characterize the nature and regulation of the Myo10-Wee1 interaction, (2) the consequence of this interaction for aspects of Wee1 function, such as its kinase activity and subcellular distribution, will be characterized via the respective use of in vitro kinase assays and various imaging techniques, including confocal 4D (4D = x, y, z, time) microscopy, and (3) the mechanisms by which the Myo10-Wee1 interaction affects spindle function will be examined via injection of mRNAs and morpholinos into embryos in order to express and deplete different proteins, respectively. Importantly, the molecular mechanisms identified in this vertebrate system are likely to be conserved in humans. PUBLIC HEALTH RELEVANCE: Irregularities in the mitotic spindle, the cellular structure that separates chromosomes during cell division, are common in a variety of tumor cells and are thought to be a major cause of tumorigenesis. It is our hope that a better understanding of the molecules and mechanisms controlling mitotic spindle function will one day translate into clinical strategies for the early identification of cells prone to irregular mitoses. Furthermore, deeper insight into the mechanisms by which an irregular spindle arises will facilitate the development of techniques for the prevention and/or treatment of improper mitotic spindles in cancer.